A MAPK signaling pathway is a common signaling pathway involved in cell growth. The MAPK signaling pathway is a protein kinase cascade composed of three kinds of kinase groups, i.e., MAPK, MAPK kinase (hereinafter abbreviated as MAPKK), and MAPKK kinase (hereinafter abbreviated as MAPKKK), and is highly conserved in eukaryotes. In mammals, the MAPK are classified into four kinds of MAPK family molecules, specifically, extracellular signal-regulated protein kinases 1 and 2 (hereinafter referred to as ERK1/2), ERK5, Jun N-terminal kinase/stress-activated protein kinase (hereinafter abbreviated as JNK/SAPK), and p38 MAPK, and are known to form cascades independent of each other. Of those MAPK family molecules, ERK1/2 and ERK5 are each independently involved in a MAPK signaling pathway that is mainly activated by stimulation with a growth factor or the like. The MAPK signaling pathway in which ERK1/2 are involved is sometimes called a canonical MAPK signaling pathway. On the other hand, JNK/SAPK and p38 MAPK are each independently involved in a novel MAPK signaling pathway that is activated by an inflammatory cytokine such as interleukin-1 (IL-1) or tumor necrosis factor-α (TNF-α), or a physicochemical stress such as irradiation with UV light or hypertonic stimulation.
In the canonical MAPK signaling pathway, cell growth and survival are promoted by phosphorylation of downstream proteins by three kinds of kinases, i.e., Raf, MAPK/ERK kinase (hereinafter abbreviated as MEK), and ERK (Non Patent Document 1). Raf is a MAPKKK having serine/threonine kinase activity, and its family includes B-Raf (hereinafter sometimes referred to as BRAF), Raf-1, A-Raf, and the like. Raf is activated by Ras and operates the MAPK signaling pathway. MEK is a MAPKK having functions of phosphorylating not only a tyrosine residue but also a serine residue and a threonine residue, and is activated by phosphorylation by Raf and specifically phosphorylates ERK1/2. ERK is a MAPK having serine/threonine kinase activity. It is known that ERK1 and ERK2 having extremely high homology are present. ERK1/2 are phosphorylated by MEK1 and MEK2 (hereinafter referred to as MEK1/2).
In the canonical MAPK signaling pathway, when a growth factor such as epidermal growth factor (hereinafter abbreviated as EGF) binds to a receptor having tyrosine kinase activity on a cell membrane, a receptor tyrosine kinase (hereinafter sometimes abbreviated as RTK) is dimerized and activated (Non Patent Document 2). When the RTK is autophosphorylated, an adaptor protein Grb2 binds thereto. Grb2, to which a guanine nucleotide exchange factor, son of sevenless (SOS), is bound, promotes a guanosine diphosphate/guanosine triphosphate (hereinafter abbreviated as GDP/GTP) exchange reaction of a G protein, Ras (there are known K-ras, H-ras, and N-ras) (Non Patent Document 3). Then, Ras is activated, which leads to activation of Raf serine/threonine kinase. Raf directly phosphorylates MEK1/2, and phosphorylated MEK1/2 phosphorylate ERK1/2. Finally, phosphorylated ERK1/2 enter the nucleus and activate transcription of Elk-1 or cyclin D1, resulting in cell growth.
There are many reports on mutations and overexpression of factors involved in the MAPK signaling pathway in tumor cells. The overexpression and mutations of the receptor tyrosine kinase such as EGF receptor (hereinafter abbreviated as EGFR) or Her2 have been reported, which result in abnormal activation of the MAPK signaling pathway leading to malignant transformation (Non Patent Documents 4 to 6). In particular, the overexpression and mutations of EGFR are found in 50% or more of human malignant tumors. An active mutation of Ras is found in 30% of all malignant tumors, and in particular, is found in 90% of pancreatic cancers and 50% of colorectal cancers (Non Patent Documents 7 to 9). Similarly, an active mutation of BRAF is found in 63% of malignant melanomas, 45% of thyroid cancers, and 36% of ovary cancers (Non Patent Documents 10 to 12). An active mutation of BRAF is caused by constitutive activation of a part having a catalytic action through its structural change due to a substitution of a valine residue to a glutamic acid residue (V600E) in an amino acid residue at position 600, which is an active part. As a result, a downstream factor is activated without stimulation with a growth factor or the like, and hence cells abnormally grow, leading to malignant transformation (Non Patent Document 13).
MEK is positioned downstream of Ras and Raf and has high substrate specificity, and ERK as its substrate is activated in many types of tumor cells. Therefore, an inhibitor that targets MEK has been developed for the purpose of suppressing cell growth (Non Patent Document 14).
The MEK inhibitor developed for the first time is PD098059 (Parke-Davis). This compound exhibited inhibitory activity on MEK with an IC50 value of about 10 μmol/L. Next, U0126 (formerly, DuPont Pharma) was developed. U0126 inhibited MEK1/2 with an IC50 value of from about 5 to 7 nmol/L. PD098059 and U0126 exhibited growth-suppressive activity in vitro, but was not subjected to a clinical trial (Non Patent Documents 15 and 16).
The MEK inhibitor PD184352 (CI-1040, Parke-Davis) was reported for the first time as exhibiting a growth-suppressing effect in vivo and was subjected to a clinical trial. This compound was improved in both selectivity and inhibitory activity as compared to PD098059, and inhibited MEK1 in a non-adenosine triphosphate (ATP)-competitive manner with an IC50 value of 17 nmol/L. Further, at a preclinical stage, cell growth inhibitory activities on colorectal cancer cells and malignant melanoma were confirmed (Non Patent Document 17). PD0325901 (Pfizer) and AZD6244 (AstraZeneca/Array BioPharma) were developed as analogous compounds of PD184352. PD0325901 inhibited MEK1/2 in a non-ATP-competitive manner with an IC50 value of about 1 nmol/L, and exhibited more potent growth-suppressive activity than PD184352 in vivo (Non Patent Document 18). In a clinical trial, an antitumor effect and a decrease in phosphorylation of ERK were found in a phase I clinical trial and a phase II clinical trial (Non Patent Documents 19 and 20). AZD6244 inhibits MEK in a non-ATP-competitive manner with an IC50 value of about 12 nmol/L (Non Patent Document 21). This compound exhibits an antitumor effect in a clinical trial and is under a clinical trial at present.
A MEK inhibitor SMK-17 (Daiichi Sankyo Company, Limited), which was developed directed toward potent MEK inhibitory activity and excellent pharmacokinetics, has been found to have MEK1/2-specific inhibitory activity and growth-suppressive activity (Non Patent Document 22 and Patent Document 1).
An another signaling pathway, a Wnt/β-catenin signaling pathway, is known to involve in organism's development, cell growth, and oncogenesis. In the Wnt/β-catenin signaling pathway under a state in which a ligand Wnt does not act, a cancer-suppressing protein adenomatous polyposis coli (APC), a scaffold protein Axin (Non Patent Documents 23 to 25), glycogen synthase kinase-3 (hereinafter abbreviated as GSK-3), and casein kinase 1 (abbreviated as CK1) form a complex with β-catenin (hereinafter sometimes abbreviated as β-cat), in which β-catenin is phosphorylated by GSK-3 (Non Patent Document 23 and Non Patent Document 26) or CK1 (Non Patent Documents 27 to 29). Phosphorylated β-catenin is degraded via a ubiquitin-proteasome pathway (Non Patent Documents 30 to 32), and hence β-catenin is suppressed to a low expression level. However, when Wnt binds to a complex of a transmembrane receptor Frizzled (hereinafter abbreviated as Fz) and its coupled receptor LRP (Fz/LRP complex), Dishevelled is phosphorylated and phosphorylation activity of GSK-3 is inhibited via Axin (Non Patent Documents 33 and 34). This inhibits the phosphorylation of β-catenin. As a result, β-catenin is stored in the cytoplasm without being degraded (Non Patent Document 35). After that, β-catenin enters the nucleus and forms a complex with a transcription factor T-cell factor (hereinafter abbreviated as TCF) (Non Patent Documents 36 and 37). Finally, transcriptional activation of a target gene such as c-myc, which is involved in cell growth, survival, and apoptosis, or cyclin D1, which promotes cell growth, is caused.
Mutations of APC and β-catenin in tumor cells, which are constituent factors of the Wnt/β-catenin signaling pathway, have been reported. These mutations are found in 90% of colorectal cancers (Non Patent Documents 38 and 39). The mutations of APC are, in most cases, mutations deficient in binding sites for Axin and β-catenin (Non Patent Document 40). This results in that a complex that phosphorylates β-catenin is not formed, and β-catenin enters the nucleus without being phosphorylated and constitutively activates the Wnt/β-catenin signaling pathway. Active mutations among the mutations of β-catenin are, in most cases, substitution mutations of amino acid residues of a phosphorylation site by GSK-3, for example, a serine residue at position 33 (S33), serine residue at position 37 (S37), and threonine residue at position 41 (T41) thereof, and an amino acid residue of a phosphorylation site by CK1, for example, a serine residue at position 45 (S45) thereof, to an amino acid residue not phosphorylated by GSK-3 or CK1. This results in that β-catenin having an active mutation enters the nucleus without being phosphorylated by the above-mentioned complex and constitutively activates the Wnt/β-catenin signaling pathway, resulting in canceration of cells (Non Patent Document 39).